Introduction

In gram-negative bacteria, ammonia is a unique molecule required for nitrogen anabolism. Nitrogen is essential for the synthesis of key constituents of the cell, such as amino acids, NAD, pyrimidines, purines, and amino sugars1. In general, ammonia assimilation in bacteria is associated with three enzymes: glutamine synthetase (GS), glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH)2. Nitrogen starvation leads to GS-GOGAT mediated glutamine synthesis, whereas high ammonia availability leads to the activation of GDH3,4. The pathways involved in ammonia assimilation are common among bacteria, cyanobacteria, algae, yeasts, and fungi5. In Helicobacter pylori, ammonia is produced by urease, which exhibits higher activity compared with that in other species, and is used in the neutralization of gastric acid for survival in animal hosts6. The remaining ammonia is then processed by GS. The GS in H. pylori (Hpy GS) is a critical enzyme that is involved in processing the ammonia released by urease activity, and nitrogen metabolism7.

GS is an enzyme involved in the biosynthetic reaction producing glutamine from the condensation of glutamate and ammonia8. This enzyme is present in all living organisms and is a large homo-oligomeric complex composed of eight, ten, or twelve subunits assembled into two face-to-face rings9. The catalytic and regulatory loops of all GS enzymes including Hpy GS are well conserved, resulting in a similar biosynthetic mechanism. Each active site at the subunit-subunit interface has two main entrances for ATP and glutamate10. The active site of GS is generally described as a ‘bifunnel’, where the entrance for ATP is located at the top of the bifunnel opening to the external surface, and glutamate can enter through the bottom of the bifunnel. With the presence of divalent cations such as magnesium or manganese ions, the carboxyl oxygen in the side chain of glutamate attacks the terminal phosphorus of ATP, resulting in a γ-glutamyl phosphate intermediate. The intermediate is subsequently attacked by ammonia to form glutamine11,12.

The GS in all living organisms can be categorized into three different classes: GS I, GS II, and GS III13. GS I and GS III enzymes are found in bacteria and archaea and mostly form a dodecamer, whereas GS II enzymes are found in eukaryotes and form a decamer14,15. GS I enzymes are further subdivided into two GS isoenzymes: GS I-α and GS I-β. GS I-α enzymes are generally found in low G + C gram-positive bacteria, thermophilic bacteria, and euryarchaeota, whereas GS I-β enzymes are found in other bacteria species16,17. There are two distinct differences between GS I-α and GS I-β enzymes. GS I-α enzymes are mainly feedback-inhibited by the end products of glutamine metabolism including AMP and glutamine, whereas GS I-β enzymes are additionally inactivated by the adenylation of a tyrosine residue near the active site (NLYDLP). Furthermore, the insertion of a specific 25-amino acid residues (residues 146–170 in Salmonella typhimurium GS) occurs in GS I-β but not GS I-α13. However, Hpy GS is unique and does not fit into either subdivision. Although the structural and sequence information of Hpy GS indicate its relationship with GS I-β due to the presence of a specific 25-amino acid residues (residues 155–179), the adenylation site (NLYDLP) found in similar homologs is replaced in Hpy GS (NLF407KLT), suggesting a lack of the adenylation regulatory mechanism in Hpy GS.

Several crystal structures of GS from bacterial species including Mycobacterium tuberculosis, Salmonella enterica, Bacillus subtilis, and Bacteroides fragilis have been determined18,19,20,21. The GS from B. subtilis (Bsu GS) is categorized as GS I-α, which lacks both the adenylation regulatory mechanism and the specific 25-amino acid residues22,23. Structural analysis of Bsu GS has revealed that it has a unique feedback inhibition mechanism distinct from that of GS I-β enzymes20. The GS from S. typhimurium (Sty GS), which is categorized as GS I-β, is subjected to adenylation on the tyrosine residue in NLY407DLP and has the specific 25-amino acid residues24. The crystal structures of Sty GS reports that a flexible loop (residues 324–329 in S. typhimurium) near the active site protects the intermediate and completes the ammonium binding site upon ATP and glutamate binding12.

Hpy GS is a critical enzyme required for the survival of H. pylori in animal hosts and may be used to target H. pylori, which causes duodenal ulcers and stomach cancer in humans7. Structural information on Hpy GS will be valuable for designing new selective inhibitors. We performed crystallographic analysis to further understand the structural and regulatory features of Hpy GS, which cannot be classified as either GS I-α or GS I-β. Here, we report the Hpy GS structures of the apo-, substrate-bound form, and the intermediate state. The results demonstrated the formation of Hpy GS from a dimer of hexameric rings, and a comparison of the apo-, substrate-bound form, and intermediate state revealed the catalytic mechanism upon substrate binding.

Results

Structure determination and model quality

To determine the structure of Hpy GS, crystals of the apo- and substrate-bound forms in complex with ATP and phosphinothricin (PPT) were obtained. The apo structure of Hpy GS (Hpy GSapo) was determined at 2.8 Å by molecular replacement using the GS model from S. typhimurium (1F52) and refined to crystallographic Rwork and Rfree values of 18.99% and 26.01%, respectively. The refined model (PDB entry 5ZLI) contained 2,846 amino acid residues of six identical subunits in the asymmetric unit, which could be used to generate a dodecamer. In each subunit, N-terminal residues (Met1–Asn7 in subunit A, E, and F; Met1–Gln6 in subunit B, C, and D) and internal residues (Leu408–Gly418 in all subunits) were disordered.

The substrate-bound form of Hpy GS (Hpy GSsub) was obtained in the presence of ATP and PPT, which is a structural analogue of glutamate. Hpy GSsub was determined at 2.9 Å and refined to crystallographic Rwork and Rfree values of 16.45% and 24.27%, respectively. The refined model (PDB entry 5ZLP) contained 5,704 amino acid residues of twelve identical subunits in the asymmetric unit. In each subunit, N-terminal residues (Met1–Thr5 in subunit A, D, G, and L; Met1–Gln6 in subunit B, F, H, and K; Met1–Asn7 in subunit C; Met1–Ser8 in subunit E; Met1–Ile2 in subunit I; Met1–Val3 in subunit J) and internal residues (Leu408–Gly418 in all subunit) were disordered. Model qualities and refinement statistics are summarized in Table 1.

Table 1 Data collection and refinement statistics.
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Overall structure of Hpy GS

The monomeric structure of Hpy GS containing 481 amino acid residues was composed of 14 α-helices and 15 β-strands, which could be divided into the N-terminal domain (residues 1–113) and C-terminal domain (residues 114–481) (Fig. 1a). The N-terminal domain was exposed to the solvent, whereas the C-terminal helix, called the ‘helical thong’, was inserted into a hydrophobic hole in the opposite subunit of the other hexameric ring (Fig. 1b). The catalytic and regulatory loops of GS were highly conserved, which included the Glu loop (flap, PGYE337AP), Asp loop (latch, D60–D74), Asn loop (residues F265–N274), Tyr loop (S163–Y190–M199), and adenylation loop (NLF407KLT)9,12 (Fig. 2a,b). These loops completed the classical ‘bifunnel’ structure of the active site. In general, the adenylation loop is located near the active site and regulates GS activity by adenylation13. Although the location of the adenylation loop of Hpy GS was conserved with that of other GS enzymes, the tyrosine residue (NLYDLP) of the adenylation site was replaced with phenylalanine (NLF407KLT) (Fig. 2a). As a result, Hpy GS could not be regulated by adenylation which is a unique feature among GS I-β subfamily members.

Figure 1
figure 1

Overall structure of GS from H. pylori. (a) The monomeric structure of Hpy GSapo. Each active site has an entrance for ATP and glutamate. (b) Oligomeric structure of Hpy GSapo was generated by two-fold symmetry from hexamer in an asymmetric unit. The C-terminal helical thong of each subunit interacted with the C-terminal thong of opposite subunits. Twelve active sites were positioned at each subunit-subunit interface.

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Figure 2
figure 2

Catalytic and regulatory loops of Hpy GS. (a) Multiple sequence alignment of Hpy GS with other homologous GS sequences in bacteria. The highly conserved residues are indicated by black boxes. The active site and catalytic residues are indicated by blue boxes. The tyrosine residue replaced to phenylalanine in Hpy GS is coloured in red box. (b) The catalytic and regulatory loops were well conserved, completing the active site between the subunit-subunit interfaces. The Asp loop was contributed from the neighbouring subunit.

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Oligomeric structure of Hpy GS

Although a hexamer of Hpy GSapo was present in each asymmetric unit of the crystal, it could be used to generate a dodecamer with 62 symmetry by stacking two hexameric rings face-to-face (Fig. 1b). The hexameric structure of Hpy GS was formed mainly by the Tyr loop of each subunit interacting through hydrogen bonds, hydrophobic interactions, and salt bridges. The solvent-accessible surface area (SAS) buried at the interface between the subunits in the hexameric structure of Hpy GSapo and Hpy GSsub were calculated to 3,698 Å2 and 3,672 Å2 (~18% of the monomer surface area), respectively (Protein–Protein Interaction Server PDBePISA at http://www.ebi.ac.uk/msd-srv/prot_int/). The hexameric interface of the dodecamer was composed of C-terminal helices 13 and 14 (residues 461–481), which extended to a hydrophobic hole of the opposite hexameric ring. The C-terminal helices 13 and 14 of one hexamer was interlocked with the C-terminal helices of the opposite hexamer via hydrophobic interactions (A461, F464, P465, W466, P472, F473, F475, and I476) and hydrogen bonds (E462 Oε1 to T478 Oγ1, K469 O to H471 Nε2, P470 O to H471 Nε2, H471 Nε2 to K469 O, H471 Nε2 to P470 O, E474 Oε1 to T326 Oγ1, E474 Oε1 to K330 Nζ, E474 Oε2 to N327 Nδ2, T477 O to K35 Nζ, T478 Oγ1 to E462 Oε2, Y479 OH to T262 Oγ1, Y479 OH to A263 N, S480 O to L253 Nζ, and C481 Sγ to S373 O) (Fig. S1). The SAS buried at the interface between the hexameric rings in the dodecameric structure of Hpy GSapo and Hpy GSsub were calculated to 2,875 Å2 and 2,870 Å2 (~13% of the monomer surface area), respectively. These results demonstrated the dodecameric form of Hpy GS.

Structural comparisons

Six subunits in the asymmetric unit of Hpy GSapo were almost identical to each other with root-mean-square deviation (r.m.s.d.) values of 0.22–0.27 Å for 474 Cα atoms in residues 8–481. In Hpy GSsub, twelve subunits in the asymmetric unit were also highly similar with r.m.s.d. values of 0.29–0.36 Å for 475 Cα atoms in residues 7–481. Superposition of Hpy GSapo and Hpy GSsub gave a r.m.s.d. value of 0.50 Å for 473 Cα residues (residues 9–481), and a r.m.s.d. value of 0.50 Å for 4,742 Cα atoms for dodecamer-dodecamer comparisons. The r.m.s.d. values of the catalytic and regulatory loops were calculated and ranged from 0.45 Å to 1.1 Å (Table S1.). The adenylation loops had higher r.m.s.d. values, which may be attributed to the disorder of the loop. These results indicated that Hpy GS does not undergo a large conformational change upon substrate binding.

Active site of Hpy GS

The active site and substrate-binding residues of Hpy GS were examined using ATP and PPT as substrates. The active site was located at the centre of the bifunnel contributed by the Glu loop (flap, residues 334–339), Asn loop (residues 265–277), and Tyr loop (residues 163–199) of each subunit and the Asp loop (latch, residues 60–74) of the neighboring subunit (Fig. 2b). As shown in other GS structures, the entrance for ATP was located at the top of the bifunnel opening to the external surface, and glutamate could enter through the bottom of the bifunnel facing the hexameric interface (Fig. 1a). In general, PPT is phosphorylated in GS in the presence of ATP and forms an intermediate state analogue, phosphinothricin phosphate (P3P), and ADP24,25. However, although ATP and PPT were added to Hpy GS, ATP/ADP substrates were clearly identified in all subunits, whereas PPT/P3P substrates were not identified in most of the subunits of Hpy GS. Among twelve subunits, PPT was not phosphorylated, resulting in a substrate-bound form with PPT and ATP in three subunits. In only one subunit, PPT was phosphorylated by ATP hydrolysis, resulting in an intermediate state with P3P and ADP (Fig. 3a). Thereafter, we described the Hpy GS bound to P3P and ADP as intermediate state (Hpy GSint).

Figure 3
figure 3

Active site of Hpy GS. (a) The active site of Hpy GSsub with ATP and PPT. (b) The active site of Hpy GSint with ADP and P3P. The N, H, and O atoms of the ligands are coloured in blue, grey, and red, respectively. The C atoms of Hpy GS, ATP/ADP, and PPT/P3P are presented in light blue, green, and yellow, respectively. The 2Fo-Fc maps of ATP/ADP and PPT/P3P are shown.

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All known 19 active site and catalytic residues in bacterial GS9 were mostly well conserved in Hpy GS except for a serine residue (S53 in S. typhimurium and S57 in M. tuberculosis), which was replaced with cysteine (C63 in H. pylori). This suggests the similarity of the overall catalytic mechanism of the Hpy GS with that of other bacterial GS proteins. The adenine ring of ATP/ADP was oriented similarly to Mtb GS26, bound by hydrogen bonds (N1 to S283 Oγ, and N6 to N362 O), and stacked in the hydrophobic patch by residues F235 and R365. The phosphate group of ATP/ADP was bound by ionic interactions (O1α to R365 NH2, and O2β to R349 NH2). PPT was bound by six hydrogen bonds with Hpy GS (NP to E141 Oε1/G275 O, OTP to R331 Nε, OP to H279 Nε2/R331 NH2, and OεB to R369 NH2), and P3P was bound by seven hydrogen bonds, where the O13 atom additionally formed a hydrogen bond with R349 NH2 (Fig. 3a,b). The possible ammonium binding site was blocked by the methyl group of P3P, acting as an inhibitor.

Two or three divalent metal ions have been reported to be involved in the active site of GS in other organisms. Two metal ions (n1 and n2 metal-binding sites) were identified per subunit in the substrate-bound GS from S. typhimurium, and three metal ions (n1, n2, and n3 metal-binding sites) were identified in the substrate-bound GS from M. tuberculosis. In both the Hpy GSint and Hpy GSsub structures, one magnesium ion was identified in the n1 metal binding site. The magnesium ion was coordinated with P3P (OεB and O15), E141 (Oε1), E230 (Oε2), and E223 (Oε1) (Fig. S2).

Comparison with other GS proteins

The structural similarity of Hpy GS with other known bacterial GS proteins was determined by DALI server27. The most similar structure was the Mtb GS (GS I-β, 3ZXR) with a r.m.s.d. value of 1.2 Å for 476 equivalent Cα atoms and a Z-score of 54.3. The Sty GS (GS I-β, 1FPY) was the next most similar structure with a r.m.s.d. value of 1.2 Å for 467 equivalent Cα atoms and a Z-score of 53.9. A comparison of Hpy GS with the Bsu GS (GS I-α, 4LNI) revealed a r.m.s.d. value of 1.6 Å for 432 equivalent Cα atoms and a Z-score of 48.2. The overall structural comparison of Hpy GS with GS I-α and GS I-β revealed high structural similarities (Fig. S3).

Earlier studies of the GS from S. typhimurium, M. tuberculosis, and B. subtilis have demonstrated a structural change between the apo- and substrate-bound forms. The Glu flap in these GS structures (residues 324–329 in S. typhimurium, 324–329 in M. tuberculosis, and 301–306 in B. subtilis) was a flexible loop, which contributed to the entrance of the glutamate binding site with a conformational change between an open and closed form12,19. However, the Glu flap in both the Hpy GSapo and Hpy GSsub structures showed a similar closed conformation with little differences. The side chain of E337 in the Hpy GSapo structure was disordered, demonstrating the flexibility of the Glu flap, whereas the side chain of E337 in the Hpy GSsub structure formed strong hydrogen bonds with the N274 Oδ1 and D60 Nδ2 of the adjacent subunit (Fig. 4a,b). This finding suggests that the Glu flap in Hpy GSapo could exist in the open or closed form in the absence of substrates.

Figure 4
figure 4

Entrance for glutamate in Hpy GS. (a,b) Show the entrance for glutamate in Hpy GSapo and Hpy GSint, respectively. The N, H, and O atoms of the ligands are coloured in blue, grey, and red, respectively. ADP and P3P are represented in green and yellow, respectively. Black dotted lines denote hydrogen bonds.

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Conclusions

We determined the apo- and substrate-bound forms of Hpy GS by X-ray crystallography. A comparison of Hpy GS with Sty GS, Mtb GS, and Bsu GS also revealed high similarities in the structure and regulatory amino acid residues. However, the open form of GS was not found in Hpy GS, and the Glu flap was well ordered with a closed conformation in Hpy GSapo in the absence of substrates. This suggests that Hpy GS could exist in either the open or closed form regardless of the presence of substrates. Based on the sequence and structural information of Hpy GS, this protein cannot be clearly classified as GS I-α or GS I-β. Therefore, we may classify Hpy GS as a new member of the GS I subfamily, which has a specific 25-amino acid sequence and lacks the adenylation regulatory mechanism. As GS is a critical enzyme required for the survival of H. pylori, structural studies of Hpy GS can provide further insight into drug development for the treatment of H. pylori infection.

Methods

Expression and purification

The gene encoding GS (glnA) was amplified by polymerase chain reaction using the genomic DNA of H. pylori as a template. It was inserted into the Nde1/Xho1-digested expression vector pET28b(+) (Novagen, Germany), containing a hexahistidine tag at its N-terminus. The recombinant Hpy GS was transformed and expressed in E.coli BL21 (DE3) star pLysS cells (Invitrogen, USA). The cells harbouring the glnA gene were grown at 310 K to an OD600 of ~0.5 in Luria-Bertani medium supplemented with 30 µg mL−1 kanamycin and chloramphenicol. Overexpression of the recombinant protein was induced with 1.0 mM isopropyl β-D-thiogalactopyranoside (IPTG), and cell growth was continued at 303 K for 4 h. The cells were harvested by centrifugation at 4,200 g for 10 min at 277 K and immediately frozen at 193 K. Cell pellets were resuspended in lysis buffer [20 mM Tris–HCl pH 8.0/0.5 M NaCl/10% (v/v) glycerol/1 mM phenylmethylsulfonylfluoride] and lysed using an ultra sonicator (SonicsTM Vibra Cell VCX 750; Sonics, USA). The insoluble fractions were removed by centrifugation at 31,000 g for 1 h at 277 K.

The recombinant Hpy GS in the supernatant fraction was loaded on a nickel-charged His-trap immobilized metal affinity chromatography (IMAC) column (GE Healthcare, UK). The hexahistidine tagged Hpy GS was loaded with buffer A [20 mM Tris-HCl pH 8.0/500 mM NaCl/10% (v/v) glycerol] and eluted by buffer B [20 mM Tris-HCl pH 8.0/500 mM NaCl/10% (v/v) glycerol/300 mM imidazole]. The eluted proteins were loaded in a Superdex 200 gel-filtration column (GE Healthcare, UK) for further purification with elution buffer [20 mM Tris-HCl pH 8.0/200 mM NaCl/5% (v/v) glycerol/1 mM dithiothreitol (DTT)/2 mM MgCl2].

Crystallization

Purified Hpy GS was concentrated to 41 mg mL−1 using Centricon YM-10 (Millipore, USA) for crystallization trials. Initial screening was performed by the sitting-drop vapour diffusion method using 96-well CrystalQuick plates (Greiner Bio-One, Germany) with various commercial screens (Hampton Research, USA; Qiagen, Germany; Axygen, USA; Emerald Biosystems, USA). Each sitting drop was prepared by mixing 0.75 µl protein solution and 0.75 µl reservoir solution and incubated at room temperature. The initial crystals of Hpy GS were grown after four weeks of incubation under several conditions with polyethylene glycol (PEG) 6,000. The crystals of Hpy GS were further optimized, and the best crystals were grown in 10% (v/v) PEG 6,000, 100 mM HEPES pH 7.0, and 50 mM choline. To obtain the cocrystals with substrates, Hpy GS was concentrated to 19 mg mL−1 and mixed with 5 mM ATP, 5 mM PPT, and 5 mM magnesium chloride before crystallization. It was stored at 277 K for 1 h. The crystals of substrate-bound Hpy GS were grown in 2.0 M sodium formate and 100 mM sodium citrate at pH 5.0.

X-ray data collection

Crystals of the apo- and substrate-bound Hpy GS were transferred to a cryoprotectant solution containing 30% PEG 6,000 and 30% glycerol in reservoir solution, respectively, and immediately flash-cooled in liquid nitrogen. X-ray diffraction data were collected at 100 K on an ADSC Quantum 315 CCD image-plate detector using synchrotron radiation on beamline 5 C of the Pohang Accelerator Laboratory, Pohang, Republic of Korea. Data were collected with 1° oscillation per image and a crystal-to-detector distance of 650 mm, and a total of 180 frames were recorded. Data were processed and scaled using the HKL-2000 program suite28.

Structure determination and refinement

The crystal structure of the apo form of Hpy GS was solved by molecular replacement using the GS structure from S. typhimurium as a template (1F52) with PHASER from the CCP4 program suite. Hpy GS in complex with ATP and PPT was solved using the apo form of Hpy GS as a template in the same program suite. Both structures were refined with REFMAC from the CCP4 program suite with intensity based twin refinement. Further refinement was carried out by the COOT and PHENIX program package. The refined model was finally evaluated by MolProbity. Data collection and refinement statistics are presented in Table 1.

Accession numbers

Coordinate and structure factors have been deposited in the Protein Data Bank (PDB): apo Hpy GS, PDB ID, 5LZI; substrate-bound Hpy GS, PDB ID, 5ZLP.